Project Summary/Abstract Disruption of immune checkpoint interactions by monoclonal antibodies has replaced chemotherapy as the standard of care for metastatic melanoma, and is likely to become mainstream treatment for diverse malignancies. Such therapies target activated T cells within the tumor to enhance antitumor activity. The most successful therapies to date disrupt the interaction between PD-1 on T cells and PD-L1 on tumors. We have developed an alpaca-derived single-domain antibody (VHH) that potently inhibits PD-L1 binding to PD-1, which we have termed VHH B3. VHH B3 augments antitumor responses in vitro and in vivo to a degree comparable to commercial monoclonal antibodies. We propose to use VHH B3 as a scaffold for attaching the cytokines IL-2 and IFN-?, both of which have known antitumor activity. In this strategy, we hypothesize that PD-L1 will serve as a broadly expressed tumor antigen leading to accumulation of the B3-cytokine fusion within the tumor microenvironment. By increasing local cytokine concentrations while simultaneously disrupting one of the key pathways used by tumors to evade antitumor responses, we predict synergistic antitumor activity. Preliminary work has established a method for generating a VHH B3 fusion to IL-2 (B3-IL2) that maintains in vitro PD-L1 binding and IL-2 activity, and appears to have activity against orthotopically transplanted pancreatic cancer. We aim to similarly generate and test B3-IFN-?. After in vitro validation, both B3-cytokine fusions will be tested alone and in combination with other immune therapies in murine models of pancreatic adenocarcinoma and melanoma. We will then analyze the immune correlates of treatment response in order to understand the activity of these fusions. The long-term goal of this research is to rationally design improved treatment protocols and combination immune therapies that enhance antitumor efficacy while minimizing systemic immune toxicities.